Wogonin ameliorates the proliferation, inflammatory response, and pyroptosis in keratinocytes via NOD‐like receptor family pyrin domain containing 3/Caspase‐1/Gasdermin‐D pathway

Abstract Background Psoriasis refers to a highly prevalent and immunologically mediated dermatosis with considerable deterioration in life quality. Wogonin, a sort of flavonoid, has been mentioned to elicit protective activities in skin diseases. However, whether Wogonin is implicated in the treatment of psoriasis and its specific mechanisms are not fully understood. Aim The present work attempted to elaborate the role of Wogonin during the process of psoriasis and to concentrate on the associated action mechanism. Methods Cell counting kit‐8 (CCK‐8) method was initially applied to assay the viability of human keratinocyte HaCaT cells treated by varying concentrations of Wogonin. To mimic psoriasis in vitro, HaCaT cells were exposed to M5 cytokines. CCK‐8 and 5‐Ethynyl‐2′‐deoxyuridine assays were adopted for the measurement of cell proliferation. Inflammatory levels were examined with enzyme‐linked immunosorbent assay. Immunofluorescence staining tested nucleotide‐binding oligomerization domain (NOD)‐like receptor family pyrin domain containing 3 (NLRP3) and Caspase‐1 expressions. Western blot examined the protein expressions of proliferation‐, inflammation‐, pyroptosis‐associated factors, and NLRP3. Results Wogonin treatment antagonized the proliferation, inflammatory response, and NLRP3/caspase‐1/Gasdermin‐D (GSDMD)‐mediated pyroptosis in M5‐challenged HaCaT cells. Besides, NLRP3 elevation partially abrogated the effects of Wogonin on M5‐induced proliferation, inflammatory response, and NLRP3/caspase‐1/GSDMD‐mediated pyroptosis in HaCaT cells. Conclusion In a word, Wogonin might exert anti‐proliferation, anti‐inflammatory and anti‐pyroptosis activities in M5‐induced cell model of psoriasis and the blockade of NLRP3/Caspase‐1/GSDMD pathway might be recognized as a potential mechanism underlying the protective mechanism of Wogonin in psoriasis, suggesting Wogonin as a prospective anti‐psoriasis drug.


| INTRODUCTION
Psoriasis is recognized as a genetically inherited spectrum of skin illness mediated by immune which is portrayed as well-demarcated erythematous plaques with adherent silvery white scales clinically. 1 The global incidence rate of psoriasis among adults is approximately 0.51% ~11.43%, which may impose considerable burden on the life quality of psoriasis patients. 2Topical treatment, targeted phototherapy, biological agents, and oral systemic medications remain primary treatment approaches for psoriasis. 3However, effective strategies to cure psoriasis remain unavailable.Currently, it is generally believed that the immune-inflammatory cascade mediated by the immune cell function and the release of inflammatory factors (e.g., tumor necrosis factor-alpha [TNF-α], interleukin-1beta [IL-1β], IL-23, and IL-12) are closely implicated in the onset and progression of psoriasis despite its inexact pathogenesis. 4,5Therefore, the development of antiinflammatory agents may represent an effective therapeutic measure for psoriasis.
Chinese herbal medicine is well established as an ideal synergistic medicine applied to the diversified human diseases attributed to reduced adverse effects. 6hinese herbal medicine has been increasingly revealed to exhibit effectiveness and safety as an adjuvant treatment of psoriasis. 7,8Scutellaria baicalensis Georgi is a common heat-clearing medicine in traditional Chinese medicine, the preparation from the root of which is widely applied to treat diarrhea, dysentery, hypertension, hemorrhaging, insomnia, inflammation, and respiratory infections in clinical settings. 9However, as a naturally occurring flavonoid compound that stems from the root extract of S. baicalensis Georgi, Wogonin has not been used in Western medicine in the form of a pure chemical.Wogonin has been supported to participate in tumors, inflammatory diseases, neurological diseases dependent on a wide range of pharmacological properties including antioxidant, anti-inflammatory, antitumor, immunomodulatory as well as neuroprotective effects. 10,11Notably, Wogonin has been underlined to suppress inflammatory cell infiltration and the production of pro-inflammatory cytokines to produce protective effects against inflammatory disorders, such as mastitis, 12 cerebral ischemia-reperfusion injury, 13 and so on.Moreover, previous literatures have introduced that Wogonin can ameliorate skin disorder through modulating the expressions of inflammationassociated genes. 14,15Nevertheless, whether Wogonin functions as an anti-inflammatory agent in psoriasis remains to be elaborated.
Inflammasomes composed of multimeric protein complexes are sensors of molecular signals in response to infectious microbes and molecules derived from host proteins. 16As the most extensively studied inflammasome, nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a pivotal mediator in the immune response and disease development. 17Importantly, NLRP3 is highly expressed in psoriasis samples and the blockers of NLRP3 pathway may be recognized as novel therapeutic targets of psoriasis through ameliorating inflammatory reaction during the disease course. 18,19Moreover, Wogonin has been supported to inactivate NLPP3 inflammasome in cerebral ischemiareperfusion injury. 13ere, our research was to illuminate the impacts of Wogonin on the progression of psoriasis and further delve into the mechanism of Wogonin related to NLRP3 inflammasome.

| Cell counting kit-8 (CCK-8)
In brief, before treatment with 50, 100, and 200 ng/mL of Wogonin with or without incubation with M5 cocktail, HaCaT cells were placed in a 96-well plate (1 × 10 4 cells/ well), each well of which was then given 10-μL CCK-8 solution (Shanghai SunBio Medical Biotechnology Co. Ltd.) for 2 h in conformity to the standard manual.A microplate reader (Molecular Devices) was employed for the determination of OD450 nm value.

| 5-Ethynyl-2′-deoxyuridine (EDU) staining
Cell proliferation was measured via the employment of EDU Cell Proliferation Image Kit (Abbkine).HaCaT cells (5 × 10 3 cells/well) subjected to 96-well plates were incubated with M5 cocktail and received treatment with 50, 100, and 200 ng/mL of Wogonin, before being labeled with 50-µM EDU for 2 h in compliance with the manufacturer's request.Following the 15 min of immobilization with 4% paraformaldehyde and 10 min of permeation with 0.3% Triton X-100, cells were conjugated to Click Reaction Buffer and Hoechst 33342.Images were taken under a fluorescence microscope (Olympus).

| Statistical analyses
GraphPad Prism 8 software (GraphPad Software, Inc.) was utilized to carry out statistical analyses.All data from three parallel repeated experiments were denoted as the mean ± standard deviation.Statistical significances were measured by using of one-way ANOVA along with Tukey's post hoc test.The significance level was p < 0.05.

| Wogonin treatment obstructs the hyperproliferation of HaCaT cells exposed to M5
To identify the role of Wogonin in M5-stimulated cellular model of psoriasis in HaCaT cells, the impacts of single Wogonin treatment on the viability of HaCaT cells were first estimated and it was noticed that HaCaT cell viability displayed no evident alternations upon treatment with increasing concentrations of Wogonin (4, 8, and 16 μM) (Figure 1A).Concurrently, as delineated by CCK-8 assay, the viability of HaCaT cells was distinctly improved when exposed to M5, which was then concentration-dependently diminished by Wogonin treatment (Figure 1B).Additionally, the experimental data from EDU staining presented that the potentiated proliferation of M5-challenged HaCaT cells was halted by treatment with Wogonin in a dose-dependent manner (Figure 1C).Western blot analysis also manifested that Wogonin markedly depleted KRT6 expression that was raised in M5-treated HaCaT cells (Figure 1D).In summary, Wogonin suppressed M5-induced HaCaT cell hyperproliferation.

| Wogonin treatment mitigates the inflammatory response in HaCaT cells exposed to M5
Inflammatory response remains a crucial event during the process of psoriasis.Subsequently, the effects of Wogonin on the inflammatory response were assessed in M5-induced psoriasis model in vitro.Through ELISA analysis, it turned out that M5 exposure resulted in the remarkable upregulation on the contents of inflammatory cytokines including IL-6, TNF-α, and IL-1β in HaCaT cells, which were then all declined by Wogonin (Figure 2A).As expected, in M5-challenged HaCaT cells, the elevated activities of inflammatory molecules that emerged as critical components in the pathogenesis of psoriasis including IL-23, IL-17, and IL-22 were all prominently lowered by Wogonin, exhibiting a concentration-dependent manner (Figure 2B).It was also discovered that Wogonin dramatically lessened the levels of pro-inflammatory chemokines including CXCL1, CXCL8, and CCL20 that were enhanced in HaCaT cells imposed by M5 exposure (Figure 2C).Also, the expressions of inflammatory mediators Cox2 and iNOS were noted to be augmented in M5-exposed HaCaT cells and be downregulated in Wogonin-treated HaCaT cells challenged with M5 (Figure 2D).Accordingly, Wogonin might protect against M5-triggered inflammatory response in HaCaT cells.

| Wogonin inactivates NLRP3 to suppress M5-elicited hyperproliferation of HaCaT cells
To determine whether the possible regulatory mechanism of Wogonin in psoriasis was associated with NLRP3/Caspase-1/GSDMD-mediated pyroptosis, NLRP3 was overexpressed.Following transfection of Ov-NLRP3, NLRP3 messenger RNA, and protein expressions were both distinctly increased in HaCaT cells (Figure 4A,B).CCK-8 and EDU assays corroborated that the inhibitory role of Wogonin in the proliferation of M5-treated HaCaT  cells was partially abolished when NLRP3 was upregulated (Figure 4C,D).This finding was also further testified by the raised KRT6 expression in the M5 + Wogonin + Ov-NLRP3 group relative to the M5 + Wogonin + Ov-NC group (Figure 4E).Conclusively, NLRP3 elevation partially reversed the influences of Wogonin on the hyperproliferation of M5-exposed HaCaT cells.

| DISCUSSION
Psoriasis is a systemic disease accompanied with complicated pathogenic mechanisms involving inflammatory disorder, genetic effects, and activated immune response.As the predominant cell type in the epidermis, keratinocytes are responsible for the formation of a physical skin barrier against environmental damages through producing antimicrobial peptides and cytokines and further attracting and activating immune cells to participate in the inflammatory mechanism in the lesions, thus playing an active role in the formation and maintenance of psoriatic lesions. 22,23When the epidermal barrier of psoriasis is destructed, keratinocytes are susceptible to diverse external harmful substances, causing cell damage or even cell death. 23Accordingly, the abnormal interaction between hyperproliferative epidermal keratinocytes and self-reactive immune cells is commonly known as a hallmark of psoriasis. 22HaCaT cells are immortalized keratinocyte cell lines widely used in scientific research.Thereafter, HaCaT cells were induced by M5 to assess aberrant keratinocyte behaviors in psoriasis.Wogonin has been previously revealed to hamper cell proliferation and invasion in skin epithelioid carcinoma 24 and exhibit potent protective properties against skin damage. 14,15,25,26However, the exact role of Wogonin in psoriasis is unclear.In the present study, HaCaT cells were exposed to M5 cytokines to mimic psoriasis in vitro.Administration with Wogonin eliminated the hyperproliferation, inflammatory response as well as pyroptosis in M5-stimulated psoriatic keratinocyte model, the protective mechanism of which might be mediated by inactivation of NLRP3.
As reported, traditional Chinese medicine S. baicalensis Georgi can ameliorate the macrophage-targeted responses and arrest keratinocyte proliferation to exert pharmaceutical efficacy in psoriasis-like lesions. 27In particular, Wang et al have disclosed that Wogonin, a main effective component of S. baicalensis Georgi, can reduce photodamage in HaCaT cells induced by ultraviolet B radiation. 28Here, the current study demonstrated that the improved viability and proliferation of HaCaT cells challenged with M5 were both diminished by Wogonin treatment in a concentration-dependent  | 11 of 14 manner.KRT6 is a member of Keratins that are known as the major components of the epithelial cytoskeleton associated with the maintenance of the structural stability and integrity of keratinocytes. 29Also, this research hinted that Wogonin downregulated the expression of KRT6, a marker for hyperplasia, in M5treated HaCaT cells.Inflammatory response remains a crucial event in the initiation and progression of psoriasis. 30Besides, the hyperproliferative keratinocytes sustain and amplify the inflammatory response via expressing cytokines. 31TNF-α, IL-1β, IL-23, IL-17, IL-22, Cox2, and iNOS are all inflammatory cytokines involved in the pathogenesis of psoriasis, 32,33 and CXCL1, CXCL8, and CCL20 are antimicrobial peptides and chemokines produced by keratinocytes. 34Notably, Wogonin has been previously unraveled to suppress proinflammatory Cox2, iNOS, TNF-α, and IL-1β expression in skin diseases. 14,15,25,35Consistently, the present experimental data proved that M5 exposure resulted in the upregulation on the contents of inflammatory cytokines including IL-6, TNF-α, IL-1β, IL-23, IL-17, IL-22 CXCL1, CXCL8, CCL20, and the expression of Cox2, iNOS in HaCaT cells, which were then all concentrationdependently declined by Wogonin.
Notably, Wogonin has been introduced to block NLRP3 inflammasome activation which emerges as a key mediator of psoriatic inflammation. 13,18,36It is well documented that the activation of NLRP3 inflammasome is essential for pyroptosis in various diseases, psoriasis is also included. 37Upon activation by a wide range of stimuli, the innate immune sensor protein NLRP3 oligomerizes and interacts with adaptor protein ASC, then recruits caspase-1 to drive the assembly of NLRP3 inflammasome, resulting in the cleavage and activation of caspase-1 in turn and further cleaving GSDMD and releasing the cleaved GSDMD-N to form membrane pore, thereby contributing to pyroptosis. 38Moreover, Wogonoside can block GSDMD-mediated pyroptosis to suppress cisplatin-stimulated cardiotoxicity. 39In addition, Zhou et al. have supported that Wogonoside hampers pyroptosis to protect against obesity-elicited lipid metabolism disorders and cardiac injury. 40In this study, it turned out that NLRP3, Caspase-1, ASC, GSDMD-N, and Cleaved-Caspase-1 expressions were both augmented in HaCaT cells upon exposure to M5, which were all depleted on account of treatment with Wogonin.Further elevation of NLRP3 partially counteracted the inhibitory role of Wogonin in M5-triggered hyperproliferation, inflammatory response, as well as pyroptosis in HaCaT cells.
Several limitations should be noted in this study.First, our study only preliminarily revealed the protective role of Wogonin in M5-stimulated psoriatic keratinocyte model in vitro and the impacts of Wogonin on the severity of psoriatic response need to be further unveiled in vivo.Second, pharmacokinetic/pharmacodynamics analysis of Wogonin is lacking here, which needs to be further performed in the future.Third, whether Wogonin targets other possible molecular signaling pathways to participate in the process of psoriasis also warrants further investigation.Besides, the possible receptors of Wogonin on HaCaT cells also need to be identified in our future study.

| CONCLUSION
All in all, Wogonin was capable of alleviating the hyperproliferation, inflammatory response as well as pyroptosis in HaCaT keratinocytes exposed to M5 cytokines, the effects of which might be related to the blockade of NLRP3/Caspase-1/GSDMD pathway.This finding implied that Wogonin might serve as an attractive candidate for future development as an antipsoriatic agent.
MA ET AL.